An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A), is intimately involved in eukaryotic cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: 1) deoxyhypusine synthesis and 2) deoxyhypusine hydroxylation. Deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. We have purified this enzyme from rat testis, identified its gene in the yeast Saccharomyces cerevisiae and cloned the human cDNA. Inactivation of the deoxyhypusine synthase gene in yeast causes loss of cell viability, indicating that the hypusine modification is vital for in vivo eIF-5A activity and cell proliferation. We have characterized the physical and catalytic properties and the reaction mechanism of the enzyme. We have identified an active site residue (Lys329) of the human enzyme that is involved in enzyme-intermediate formation, and thereby is critical for catalysis. X-ray crystallography (in collaboration with Drs. D.-I. Liao and D. R. Davies) of human deoxyhypusine synthase in a complex with NAD has revealed its tetrameric structure and the NAD binding site. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti- proliferative agents.